RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete yeast and opportunistic human pa thogen of increasing clinical importance due to the increasing population o f immunocompromised patients. To further investigate signal transduction ca scades regulating fungal pathogenesis, we have identified the gene encoding a RAS homologue in this organism. The RAS1 gene was disrupted by transform ation and homologous recombination. The resulting ras1 mutant strain was vi able, but failed to grow at 37 degrees C, and exhibited significant defects in mating and agar adherence. The ras1 mutant strain was also avirulent in an animal model of cryptococcal meningitis. Reintroduction of the wild-typ e RAS1 gene complemented these ras1 mutant phenotypes and restored virulenc e in animals. A dominantly active RAS1 mutant allele, RAS1(Q67L), induced a differentiation phenotype known as haploid fruiting, which involves filame ntation, agar invasion and sporulation in response to nitrogen deprivation. The ras1 mutant mating defect was suppressed by overexpression of MAP kina se signalling elements and partially suppressed by exogenous cAMP. Addition ally, cAMP also suppressed the agar adherence defect of the ras1 mutant. Ho wever, the ability of the ras1 mutant strain to grow at elevated temperatur e was not restored by cAMP or MAP kinase overexpression. Our findings suppo rt a model in which RAS1 signals in C. neoformans through cAMP-dependent, M AP kinase, and RAS-specific signalling cascades to regulate mating and fila mentation, as well as growth at high temperature which is necessary for mai ntenance of infection.